scholarly journals Reduction of Autofluorescence in Whole Adult Worms of Schistosoma Japonicum for Immunofluorescence Assay

2021 ◽  
Author(s):  
Qunfeng Wu ◽  
Zheng Feng ◽  
Wei Hu
Keyword(s):  
Schistosoma Japonicum ◽  
Trypan Blue ◽  
Treatment Time ◽  
Female Worm ◽  
Immunofluorescence Assay ◽  
Common Phenomenon ◽  
Background Signal ◽  
Chemical Reagents ◽  
Sudan Black B ◽  
Optimized Conditions

Abstract Background Immunofluorescence assay is one of methods to understand the spatial biology by visualizing localization of biomolecules in cells and tissues. Autofluorescence, as a common phenomenon in organisms, is a background signal interfering the immunolocalization assay of schistosome biomolecules, and may lead to misinterpretation of the biomolecular function. However, applicable method for reducing the autofluorescence in Schistosoma remains unclear. Methods In order to find a suitable method for reducing autofluorescence of schistosomes, different chemical reagents, such as Sudan black B (SBB), trypan blue (TB), copper sulfate (CuSO4), Tris-glycine (Gly), and ammonia/ethanol (AE), at different concentrations and treatment time were tested, and SBB and CuSO4 were verified for the effect of blocking autofluorescence in immunofluorescence to localize the target with anti-SjCRT antibody. Results By comparing the autofluorescence characteristics of different conditions, it was found that SBB, TB and CuSO4 had a certain degree of reducing autofluorescence effect, and the best effect in females was using 50 mM CuSO4 for 6 h and in males was 0.5% SBB for 6 h. Furthermore, we have applied the optimized conditions to the immunofluorescence of SjCRT protein, and the results revealed that the immunofluorescence signal of SjCRT was clearly visible without autofluorescence interference. Conclusions We present an effective method to reduce autofluorescence in male and female worm of Schistosoma japonicum for immunofluorescence assay, which could be helpful to better understand biomolecular functions. Our method provides an idea for immunofluorescence assay in other flukes with autofluoresence.

scholarly journals Reduction of autofluorescence in whole adult worms of Schistosoma japonicum for immunofluorescence assay

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Qunfeng Wu ◽  
Zheng Feng ◽  
Wei Hu
Keyword(s):  
Schistosoma Japonicum ◽  
Trypan Blue ◽  
Treatment Time ◽  
Female Worm ◽  
Immunofluorescence Assay ◽  
Common Phenomenon ◽  
Background Signal ◽  
Chemical Reagents ◽  
Sudan Black B ◽  
Optimized Conditions

Abstract Immunofluorescence assay is one of methods to understand the spatial biology by visualizing localization of biomolecules in cells and tissues. Autofluorescence, as a common phenomenon in organisms, is a background signal interfering the immunolocalization assay of schistosome biomolecules, and may lead to misinterpretation of the biomolecular function. However, applicable method for reducing the autofluorescence in Schistosoma remains unclear. In order to find a suitable method for reducing autofluorescence of schistosomes, different chemical reagents, such as Sudan black B (SBB), trypan blue (TB), copper sulfate (CuSO4), Tris-glycine (Gly), and ammonia/ethanol (AE), at different concentrations and treatment time were tested, and SBB and CuSO4 were verified for the effect of blocking autofluorescence in immunofluorescence to localize the target with anti-SjCRT antibody. By comparing the autofluorescence characteristics of different conditions, it was found that SBB, TB and CuSO4 had a certain degree of reducing autofluorescence effect, and the best effect in females was using 50 mM CuSO4 for 6 h and in males was 0.5% SBB for 6 h. Furthermore, we have applied the optimized conditions to the immunofluorescence of SjCRT protein, and the results revealed that the immunofluorescence signal of SjCRT was clearly visible without autofluorescence interference. We present an effective method to reduce autofluorescence in male and female worm of Schistosoma japonicum for immunofluorescence assay, which could be helpful to better understand biomolecular functions. Our method provides an idea for immunofluorescence assay in other flukes with autofluoresence.

Schistosoma japonicum triose-phosphate isomerase plasmid DNA vaccine protects pigs against challenge infection

Parasitology ◽  
2005 ◽  
Vol 132 (1) ◽  
pp. 67-71 ◽  
Author(s):  
Y. ZHU ◽  
J. SI ◽  
D. A. HARN ◽  
M. XU ◽  
J. REN ◽  
...  
Keyword(s):  
Adult Worm ◽  
Schistosoma Japonicum ◽  
Dna Vaccine ◽  
Plasmid Dna ◽  
Protective Efficacy ◽  
Female Worm ◽  
Triose Phosphate Isomerase ◽  
Protective Effects ◽  
Triose Phosphate ◽  
Plasmid Dna Vaccine

The protective efficacy of a Schistosoma japonicum, Chinese strain, triose-phosphate isomerase (TPI) plasmid DNA vaccine was examined in naïve pigs. Pigs were vaccinated with the TPI DNA-plasmid alone, or in conjunction with IL-12 as pcDNA3.1-P35, pcDNA3.1-P40 plasmids via intramuscular injection. Control pigs were immunized with equivalent amounts of pcDNA3.1. Pigs were immunized 3 times at 21-day intervals and challenged 30 days after the final boost. Forty-five days post-challenge, pigs were sacrificed and perfused to compare adult worm burdens, female worm burdens, liver egg burdens and granuloma size. We found that pigs vaccinated with SjCTPI DNA alone had adult worm burdens reduced by 48·3% and that a further decrease in adult worm burdens was not seen in the group vaccinated with SjCTPI DNA in conjunction with IL-12 (46·2% reduction). The SjCTPI DNA vaccines had a more pronounced effect on reducing female worm burdens i.e. 53·6% SjCTPI alone and 59·6% for SjCTPI+IL-12. Vaccination with SjCTPI-DNA reduced liver eggs by 49·4% and this response was significantly enhanced by the addition of IL-12 (65·8% reduction in liver eggs). In addition to the dramatic protective effects seen in vaccinated pigs, we also noted that granuloma size was reduced by 42% in both groups. Thus, vaccination of pigs and other large animals in China with SjCTPI DNA vaccine will likely reduce transmission by reducing adult worm burdens and worm egg output and simultaneously reduce hepatic egg-associated pathology.

Effect of Cellulase Enzymes and Swelling Agents on Colour Strength and Colourfastness of Handloom Cotton Fabric Dyed with Butea Frondosa

2015 ◽  
Vol 19 (4) ◽  
pp. 41-47
Author(s):  
Sunita Dixit
Keyword(s):  
Sodium Hydroxide ◽  
Zinc Chloride ◽  
Copper Sulphate ◽  
Treatment Time ◽  
Strength Properties ◽  
Cellulase Enzymes ◽  
Swelling Agent ◽  
Colour Strength ◽  
Optimum Ph ◽  
Optimized Conditions

Cellulases and swelling agents are known to be effective in improving the colour strength of cotton. Nowadays, handloom fabrics, such as khadi cotton, are much more preferred due to the development of innovative designs with their use and their comfort in wearing. Also, due to increased environmental awareness, the use of natural dyes are much more preferred in the dyeing of handloom fabrics. However, khadi cotton has some major shortcomings, such as less dyeability. The present study is carried out by keeping in mind that the pretreatment of khadi cotton with cellulases, swelling agents and a combination of cellulases and swelling agents before dyeing improves the colour strength properties. Khadi cotton samples are treated with optimized conditions of the enzymes and swelling agents. The optimum pH, concentration, treatment time and temperature selected for treatment of the samples with acid cellulase enzymes are 5.5, 1.5% (owf), 45 minutes and 50°C, respectively, whereas in the case of neutral cellulase enzymes, 7.5, 2.0% (owf), 70 minutes and 70°C, respectively. The optimum concentration, treatment time and temperature selected for the treatment of the samples with sodium hydroxide, ethylenediamine and zinc chloride are 20% w/v, 60 minutes and 60°C; 80% w/v, 60 minutes and 70°C, and 80% w/v, 60 minutes and 70°C respectively. Butea frondosa dye (5 g) extracted for 75 minutes provides the best results on khadi cotton when dyeing is carried out for 90 minutes. It is observed that out of the various concentrations of mordants used with the Butea frondosa dye, the best shades of colour are obtained by using 0.04 g of alum, 0.01 g of copper sulphate, and 5 g of Babool bark. In terms of optimizing the mordanting, the best results are obtained with Butea frondosa dye when the samples are simultaneously mordanted and dyed with alum, Babool bark and alum. Pre-mordanting is selected for the copper sulphate. It is found that for all the enzyme treated (acid and neutral cellulase) as well as swelling agent treated (sodium hydroxide, ethylenediamine and zinc chloride) samples, the colour strength and colourfastness increase in comparison to the untreated samples.

Highly Efficient Regeneration Method for Analginum-Saturated Activated Carbon

2013 ◽  
Vol 781-784 ◽  
pp. 2071-2075
Author(s):  
Zhao You Zhu ◽  
Wan Ling Wang ◽  
Li Li Wang ◽  
Ying Long Wang
Keyword(s):  
Activated Carbon ◽  
Solvent Extraction ◽  
Treatment Time ◽  
Activated Carbons ◽  
Influence Factors ◽  
Optimal Conditions ◽  
Thermal Regeneration ◽  
Extraction Step ◽  
Efficient Regeneration ◽  
Optimized Conditions

The regeneration of Analginum-saturated powdered activated carbon (PAC) using solvent extraction and thermal regeneration was investigated in detail to get the optimal conditions. In the solvent extraction step, various influence factors, such as pH of solvent, treatment time, and temperature were studied respectively. In addition, the optimal conditions of thermal regeneration were determined to be 500 °C, 120 min, with blowing N2 gas at 80 mL/min. Under the optimized conditions, the regeneration efficiency of spent PAC reached as high as 95.1%. This study provides a useful reference of regeneration method for other spent activated carbons.

Mir-15a and Mir-16-1 Induce Apoptosis of Raji Cell Line

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4492-4492
Author(s):  
Chen Qin ◽  
He Dongmei
Keyword(s):  
Cell Line ◽  
Trypan Blue ◽  
Immunofluorescence Assay ◽  
Morphological Observation ◽  
Blue Dye ◽  
Human Leukemia ◽  
Double Labeling ◽  
Raji Cells ◽  
Post Transfection ◽  
Exclusion Method

Abstract MicroRNAs are endogenous small noncoding RNAs that regulate gene expression negatively at posttranscriptional level. Mir-15a and mir-16-1 are deleted or downregulated in the majority of chronic lymphomatic leukemia(CLL), which is the most common human leukemia and overexpresses the antiapoptotic B cell lymphoma2 (Bcl-2) protein. Here we investigated the effect of mir-15a and mir-16-1 on human lymhpomatic cell line Raji in induction of apoptosis. We transfected chemically synthesized mir-15a and mir-16-1 oligonucleotide into Raji cells using Lipofectamine 2000 reagent. At 24,48,72 hours after transfection, the growth inhibitory effect of Raji cells was measured by trypan blue dye exclusion method and CCK8 assay. Apoptosis was determined by morphological observation and flow cytomertry analysis after AnnexinV/PI double labeling. The expression levels of Bcl-2 protein were detected by immunofluorescence assay. Trypan blue dye exclusion method and CCK8 assay showed that transfection of mir-15a or mir-16-1 decreasesd the cell growth at 24, 48 and 72 h, which were significantly lower than those cells transfected with control oligonucleotide and untransfected cells, respectively (P<0.05). Using Hoechst staining, cells treated with mir-15a or mir-16-1 oligonucleotide displayed changes of apoptosis at 48,72h after transfection. AnnexinV/PI double dyeing assays showed early and late apoptotic cells at 48 and 72 h post-transfection. At 48,72h after transfection with mir-15a or mir-16-1 oligonucleotide, early and late apoptotic cell rate were obviously higher than untransfected cells and control miRNA group. Indirect immunofluorescence assay demonstrated that the expression of Bcl-2 was degraded at post-transfection, suggesting that Bcl-2 is a direct target of mir-15a and mir-16-1. These results show that mir-15a and mir-16-1 can induce apoptosis of human lymhpomatic cell line Raji. Our results suggest that mir-15a and mir-16-1 could be used for therapy of Bcl-2 overexpression tumors.

scholarly journals Vaccination with Calpain Induces a Th1-Biased Protective Immune Response against Schistosoma japonicum

2001 ◽  
Vol 69 (1) ◽  
pp. 386-391 ◽  
Author(s):  
Renli Zhang ◽  
Ayako Yoshida ◽  
Takashi Kumagai ◽  
Hitoshi Kawaguchi ◽  
Haruhiko Maruyama ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Schistosoma Japonicum ◽  
Egg Production ◽  
Female Worm ◽  
Large Subunit ◽  
Interleukin 4 ◽  
Histopathological Study ◽  
Spleen Cells ◽  
Enhanced Production ◽  
Ifn Γ

ABSTRACT A large subunit of calpain, a calcium-activated neutral proteinase, from Schistosoma japonicum was cloned and expressed inEscherichia coli. When BALB/c mice were immunized with purified recombinant calpain (r-calpain) emulsified in complete Freund's adjuvant, a significant reduction in the number of recovered worms and also in egg production per female worm was observed(P < 0.01). Spleen cells of the immunized mice showed enhanced production of gamma interferon (IFN-γ) by activated CD4+ T cells. Considering our observation of elevated expression of inducible nitric oxide synthase mRNA in immunized mice, r-calpain-induced IFN-γ seemed to upregulate the production of nitric oxide by macrophages and subsequently mediated the killing of schistosomulae in the lung. On the other hand, spleen cells of immunized mice showed only faint interleukin-4 production in response to r-calpain in vitro, suggesting that immunization with r-calpain alters the Th1-Th2 balance in murine hosts even during a Th2-promotingS. japonicum infection. Furthermore, histopathological study of the livers of immunized mice showed that granulomas formed around eggs were diminished in both size and number. Egg production by female worms was clearly decreased in immunized mice, suggesting that r-calpain also has antifecundity effects. Taken together, these results point to S. japonicum calpain as a potential vaccine candidate for both worm killing and disease prevention, possibly through the induction of a strong Th1-dominant environment in immunized mice.

scholarly journals Optimization of Conversion Treatment on Austenitic Stainless Steel Using Experimental Designs

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
S. El Hajjaji ◽  
C. Cros ◽  
L. Aries
Keyword(s):  
Surface Area ◽  
Surface State ◽  
Treatment Time ◽  
Conversion Coating ◽  
Real Surface ◽  
Statistical Experimental Design ◽  
High Roughness ◽  
Two Parameters ◽  
Optimized Conditions ◽  
Real Surface Area

Conversion coating is commonly used as treatment to improve the adherence of ceramics films. The conversion coating properties depend on the structure of alloy as well as on the treatment parameters. These conversion coatings must be characterized by strong interfacial adhesion, high roughness, and high real surface area, which were measured by an electrochemical method. The influence of all the elaboration factors (temperature, time, and bath composition: sulphuric acid, thiosulphate as accelerator, propargyl alcohol as inhibitor, and surface state) and also the interactions between these factors were evaluated, using statistical experimental design. The specific surface area and optical factor (α) correspond to the quantitative responses. The evaluation showed, by using a designed experimental procedure, that the most important factor was “surface state.” Sanded surface allows the formation of conversion coating with high real surface area. A further aim was to optimise two parameters: treatment time and temperature using Doehlert shell design and simplex method. The growth of the conversion coating is also influenced by treatment time and temperature. With such optimized conditions, the real surface area of conversion coating obtained was about 235 m2/m2.

scholarly journals Label-Free Capacitive Biosensor for Detection of Cryptosporidium

Sensors ◽  
2019 ◽  
Vol 19 (2) ◽  
pp. 258 ◽  
Author(s):  
George Luka ◽  
Ehsan Samiei ◽  
Soroush Dehghani ◽  
Thomas Johnson ◽  
Homayoun Najjaran ◽  
...  
Keyword(s):  
Water Samples ◽  
Fluorescein Isothiocyanate ◽  
Environmental Water ◽  
Immunofluorescence Assay ◽  
Label Free ◽  
Intestinal Protozoan ◽  
Detection Range ◽  
Cryptosporidium Oocysts ◽  
Capacitive Biosensor ◽  
Optimized Conditions

Cryptosporidium, an intestinal protozoan pathogen, is one of the leading causes of diarrhea in healthy adults and death in children. Detection of Cryptosporidium oocysts has become a high priority to prevent potential outbreaks. In this paper, a label-free interdigitated-based capacitive biosensor has been introduced for the detection of Cryptosporidium oocysts in water samples. Specific anti-Cryptosporidium monoclonal antibodies (IgG3) were covalently immobilized onto interdigitated gold electrodes as the capture probes, and bovine serum albumin was used to avoid non-specific adsorption. The immobilization of the antibodies was confirmed by measuring the change in the contact angle. The detection was achieved by measuring the relative change in the capacitive/dielectric properties due to the formation of Cryptosporidium-antibody complex. The biosensor has been tested for different concentrations of Cryptosporidium. The results show that the biosensor developed can accurately distinguish different numbers of captured cells and densities on the surface of the biosensor. The number of Cryptosporidium oocysts captured on the electrode surface was confirmed using a fluorescein isothiocyanate (FITC) immunofluorescence assay. The response from the developed biosensor has been mainly dependent on the concentration of Cryptosporidium under optimized conditions. The biosensor showed a linear detection range between 15 and 153 cells/mm2 and a detection limit of 40 cells/mm2. The label-free capacitive biosensor developed has a great potential for detecting Cryptosporidium in environmental water samples. Furthermore, under optimized conditions, this label-free biosensor can be extended for detection of other biomarkers for biomedical and environmental analyses.

scholarly journals Dose-dependent Inhibition of Gynecophoral Canal Protein Gene Expression in Vitro in the Schistosome (Schistosoma japonicum) by RNA Interference

2005 ◽  
Vol 37 (6) ◽  
pp. 386-390 ◽  
Author(s):  
Guo-Feng Cheng ◽  
Jiao-Jiao Lin ◽  
Yi Shi ◽  
You-Xin Jin ◽  
Zhi-Qiang Fu ◽  
...  
Keyword(s):  
Schistosoma Japonicum ◽  
Protein Gene ◽  
Female Worm ◽  
Transcript Level ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Male Worm ◽  
Dependent Inhibition ◽  
Dose Dependent

Abstract The gynecophoral canal protein gene SjGCP of Schistosoma japonicum that is necessary for the pairing between the male and female worms is specifically expressed in the adult male worm. This protein is widely distributed in the adult female worm after pairing. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence were employed to analyze the relationship between the RNAi effect and dsRNA dosage in the parasites. The results revealed that the inhibition of SjGCP expression by siRNA is dose-dependent. RT-PCR analysis showed that the SjGCP transcript level was reduced by 75% when 100 nM dsRNA was applied.

Optimization and Application of Liposome-Mediated Transfection of pEGFP-N1 in FRT Cells

2014 ◽  
Vol 912-914 ◽  
pp. 1953-1956
Author(s):  
Guo Yan Xiang ◽  
Yun Qiao Zhang ◽  
Yu Xuan Zang ◽  
Hang Fei Zhu ◽  
Zhong Xin Zhang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Trypan Blue ◽  
Transfection Efficiency ◽  
Fluorescent Microscopy ◽  
The Other ◽  
Trypan Blue Exclusion ◽  
Optimized Conditions

This study aimed to optimize liposome-mediatedtransfection conditions of pEGFP-N1 in FRT cells and to investigatewhether the optimized conditions were the optimal liposome-mediated transfectionconditions of the other vectors in FRT cells or not. The pEGFP-N1 were transfectedinto FRT cells, with the conditions of different cell confluence、ratio and quantity of vectors / liposome. At the sametime, pEGFP-N1-Aquaporin1、pEGFP-N1-Aquaporin3and pEGFP-N1-Aquaporin4 were transfected into FRT cells, respectively, withconditions of being same as pEGFP-N1. The inverted fluorescent microscopy was used to observe cytotoxicity and the expressionof EGFP in FRT cells. Transfection efficiency was measured by flow cytometry and cell viability was measured by trypanblue exclusion. The results showed that the expression of EGFP reachedthe highest at 36h after transfection. Flow cytometryand trypan blue exclusion tests showed when the cell confluence was 70%、the ratio and quantity of vectors / liposome was 1: 4(2.0ng: 8.0μL), pEGFP-N1 got higher transfection efficiency (46.97±0.32) % andcell viability (63.47±0.32) %. Under the same conditions, AQP1、AQP3 and AQP4 got the highest transfection efficiency and higher cell viability, too. The study would provide anexperimental evidence for efficient transfection of the other vectors in FRT cells.

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